Urea and thiourea derivatives of glycopeptides

ABSTRACT

The present invention is directed to NLEU-carbamoyl and thiocarbamoyl derivatives of A82846B and NDISACC variations thereof. These derivatives are useful as antibacterials and also as starting materials from which further antibacterial compounds are prepared.

This application claims priority to provisional application Ser. No.60/047,196 dated May 20, 1997.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to N^(LEU) -carbamoyl andthiocarbamoyl derivatives of A82846B and N^(DISACC) variations thereof.These derivatives are useful as antibacterials and also as startingmaterials from which further antibacterial compounds are prepared.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds of the formula ##STR1##wherein R¹ represents O or S; R² represents

alkyl of C₁ -C₁₀,

phenyl,

naphthyl, or

phenyl substituted by one or two substituents, each of which isindependently halo, loweralkyl of C₁ -C₄, loweralkoxy of C₁ -C₄,benzyloxy, nitro, or ##STR2## wherein each R^(2a) is independentlyloweralkyl of C₁ -C₄ ; and R³ represents hydrogen or --CH₂ --R^(3a)wherein R^(3a) represents

alkyl of C₁ -C₁₁,

alkyl of C₁ -C₁₁ --R⁴, or

R⁴ -(0.sub.(0 or 1) --R⁴)₀ or 1,

wherein each R⁴ is independently phenyl or phenyl substituted by one ortwo substituents, each of which is independently halo, loweralkyl of C₁-C₄, loweralkoxy of C₁ -C₄, or loweralkylthio of C₁ -C₄, andpharmaceutically acceptable salts thereof.

The compounds of the present invention are prepared by reacting a parentglycopeptide of the formula ##STR3## wherein R³ is as defined above,with an isocyanate or isothiocyanate of the formula R¹ CN--R², whereinR¹ and R² are as defined above. This is the first step of the so-calledEdman degradation, which is a two-step process for the cleavage of theN-terminal residue of a peptide or protein.

The reaction to prepare the present compounds is carried out in a polarsolvent, such as water, in the presence of an organic base, such aspyridine. Generally the reaction is carried out at a temperature ofabout 15° C. to about 35° C. for one to five hours. The reaction ispreferably carried out at a temperature from about 25° C. to 30° C. forone to two hours, in water with pyridine as the base. The reactionconsumes equimolar amounts of the reactants but a slight excess of theisocyanate or isothiocyanate is preferred. The product is separated andpurified if desired in conventional procedures. When it is desired, asalt can be prepared in standard procedures.

The following examples illustrate the preparation of the compounds ofthe present invention.

PREPARATION OF COMPOUND OF EXAMPLE 22

N^(DISACC) -(p-(p-Chlorophenyl)benzyl)A82846B trihydrochloride (100.0mg, 0.0526 mmol) was dissolved in 10 ml H₂ O-pyridine (1:1 v/v) andtreated with phenyl isothiocyanate (0.010 ml, 0.083 mmol). The resultingmixture was stirred at room temperature for 1 hour at which time HPLCanalysis indicated complete consumption of the starting material. Thereaction mixture was concentrated in vacuo and the crude product waspurified by preparative HPLC to give 76.6 mg (76% yield) of N^(LEU)-(phenylthiocarbamoyl)-N^(DISACC) -(p-(p-chlorophenyl)benzyl) A82846B.FAB-MS: calc. For C₉₃ H₁₀₂ Cl₃ N₁₁ O₂₆ S 1925.5, obtained 1928.5 (M+3).

PREPARATION OF COMPOUND OF EXAMPLE 23

A82846B triacetate (270 mg, 0.157 mmol) was dissolved in 30 ml H₂O-pyridine (1:1 v/v) and treated with phenyl isocyanate (0.030 ml, 0.277mmol). The resulting mixture was stirred at room temperature for 1 hourat which time HPLC analysis indicated complete consumption for thestarting material. The reaction mixture was concentrated in vacuo andthe crude product was purified by preparative HPLC to give 62.5 mg (23%yield) of N^(LEU) -(phenylcarbamoyl)-A82846B. FAB-MS: Calc. For C₈₀ H₉₃Cl₂ N₁₁ O₂₇ 1709.6, obtained 1712.1 (M+3).

The HPLC procedures reported in these examples were as follows:

Analytical: Reactions were monitored by analytical HPLC using a WatersC₁₈ μBondapak or Novapak C₁₈ column (3.9×300 mm) and UV detection at 280nm. Elution was accomplished with a linear gradient of 5% CH₃ CN-95%buffer to 80% CH₃ CN-20% buffer over 30 minutes. The buffer used was0.5% triethylamine in water, adjusted to pH 3 with H₃ PO₄.

Preparative: Crude reaction mixtures were purified by preparative HPLCusing a Waters C₁₈ Nova-Pak column (40×300 mm) and UV detection at 280nm. Elution was accomplished with a linear gradient of 5% CH₃ CN-95%buffer to 80% CH₃ CN-20% buffer over 30 minutes. The buffer used was0.5% triethylamine in water, adjusted to pH 3 with H₃ PO₄. The desiredfractions were subsequently desalted with a Waters C₁₈ Sep-Pak (35 cc)followed by lyophilization.

Compounds were desalted as follows. A Waters Sep-Pak cartridge waspre-wet with methanol (2-3 column volumes) then conditioned with water(2-3 column volumes). The sample, dissolved in a minimum volume ofwater, was loaded onto the Sep-Pak column which was then washed withwater (2-3 column volumes) to remove the unwanted salts. The product wasthen eluted with an appropriate solvent system, typically 1:1 CH₃ CN/H₂O, CH₃ CN, and/or methanol. The organic solvent component was removed invacuo and the resulting aqueous solution lyophilized to give the finalproduct.

Representative compounds of the present invention are listed in thefollowing table:

                                      TABLE I    __________________________________________________________________________    Ex                               Analytical    #  Name                 FAB-MS                                 M + X                                     HPLC, min    __________________________________________________________________________     1 N.sup.LEU -          1728.5                                 3   18.2*       (PHENYLTHIOCARBAMOYL)A82846B     2 N.sup.LEU -(PHENYLTHIOCARBAMOYL)-                            1852.3                                 3   21.4*       N.sup.DISACC -(p-CHLOROBENZYL)A82846B     3 N.sup.LEU -(PHENYLTHIOCARBAMOYL)-                            1911.0                                 3   23.6*       N.sup.DISACC -(p-PHENOXYBENZYL)-       A82846B     4 N.sup.LEU -(PHENYLTHIOCARBAMOYL)-                            1894.5                                 3   23.2*       N.sup.DISACC -(p-PHENYLBENZYL)A82846B     5 N.sup.LEU -(1-NAPHTHYLTHIOCARBAMOYL)                            1778.5                                 3   19.8*       A82846B     6 N.sup.LEU -(1-       1902.5                                 3   15.4*       NAPHTHYLTHIOCARBAMOYL)-N.sup.DISACC -       (p-CHLOROBENZYL)A82846B     7 N.sup.LEU -(1-       1960.6                                 3   17.1*       NAPHTHYLTHIOCARBAMOYL)-N.sup.DISACC -       (p-PHENOXYBENZYL)A82846B     8 N.sup.LEU -((p-CHLOROPHENYL)-                            1763.0                                 4   20.5*       THIOCARBAMOYL)A82846B     9 N.sup.LEU -((p-METHOXYPHENYL)-                            1757.3                                 2   21.0*       THIOCARBAMOYL)A82846B    10 N.sup.LEU -((p-CHLOROPHENYL)-                            1944.3                                 3   26.9*       THIOCARBAMOYL)-N.sup.DISACC -(p-       PHENOXYBENZYL)A82846B    11 N.sup.LEU -((p-METHOXYPHENYL)-                            1940.3                                 3   26.0*       THIOCARBAMOYL)-N.sup.DISACC -(p-       PHENOXYBENZYL)A82846B    12 N.sup.LEU -((p-CHLOROPHENYL)-                            1887.5                                 4   24.8*       THIOCARBAMOYL)-N.sup.DISACC -(p-       CHLOROBENZYL)A82846B    13 N.sup.LEU -((p-METHOXYPHENYL)-                            1882.5                                 3   25.2*       THIOCARBAMOYL)-N.sup.DISACC -(p-       CHLOROBENZYL)A82846B    14 N.sup.LEU -((p-NITROPHENYL)-                            1774.0                                 3   19.1*       THIOCARBAMOYL)A82846B    15 N.sup.LEU -((p-(DIMETHYLAMINO)-                            1771.4                                 3   17.6*       PHENYL)THIOCARBAMOYL)A82846B    16 N.sup.LEU -((p-(BENZYLOXY)PHENYL)-                            1834.4                                 3   23.3*       THIOCARBAMOYL)A82846B    17 N.sup.LEU -((p-n-BUTYLPHENYL)-                            1784.4                                 3   17.0*       THIOCARBAMOYL)A82846B    18 N.sup.LEU -((p-n-BUTYLPHENYL)-                            1966.5                                 3   21.4**       THIOCARBAMOYL)-N.sup.DISACC -(p-       PHENOXYBENZYL)A82846B    19 N.sup.LEU -((p-(DIMETHYLAMINO)-                            1953.3                                 3   17.1**       PHENYL)THIOCARBAMOYL)-N.sup.DISACC -       (p-PHENOXYBENZYL)A82846B    20 N.sup.LEU -((p-(BENZYLOXY)PHENYL)-                            2016.3                                 3   21.1**       THIOCARBAMOYL)-N.sup.DISACC -(p-       PHENOXYBENZYL)A82846B    21 N.sup.LEU -(PHENYLTHIOCARBAMOYL)-                            1874.6                                 3   19.0**       N.sup.DISACC -(p-n-BUTYLBENZYL)-       A82846B    22 N.sup.LEU -(PHENYLTHIOCARBAMOYL)-                            1928.5                                 3   20.3**       N.sup.DISACC -(p-(p-CHLOROPHENYL)-       BENZYL)A82846B    23 N.sup.LEU -(PHENYLCARBAMOYL)A82846B                            1712.1                                 3   13.8**    24 N.sup.LEU -(PHENYLCARBAMOYL)-N.sup.DISACC -                            1894.2                                 3   18.9**       (p-PHENOXYBENZYL)A82846B    25 N.sup.LEU -(n-DECYLTHIOCARBAMOYL)-                            1792.4                                 3   N.A.       A82846B    __________________________________________________________________________     *Waters C.sub.18 NovaPak column     **Waters C.sub.18 μBondapak

The compounds of the present invention are useful for the treatment ofbacterial infections. Therefore, in another embodiment, the presentinvention is directed to a method for controlling a bacterial infectionin a host animal, typically a warm-blooded animal, which comprisesadministering to the host animal an effective, antibacterial amount of acompound of the present invention. In this embodiment, the compounds canbe used to control and treat infections due to various bacteria, butespecially gram-positive bacteria. In a preferred embodiment, thecompounds are used to control and treat infections due to bacteriaresistant to existing antibacterials. For example, certain bacteria areresistant to methicillin, and yet others are resistant to vancomycinand/or teicoplanin. The present compounds provide a technique forcontrolling and treating infections due to such resistant bacterialspecies.

In carrying out this embodiment of the invention, the compounds of thepresent invention can be administered by any of the conventionaltechniques, including the oral route and parenteral routes such asintravenous and intramuscular. The amount of compound to be employed isnot critical and will vary depending on the particular compoundemployed, the route of administration, the severity of the infection,the interval between dosings, and other factors known to those skilledin the art. In general, a dose of from about 0.5 to about 100 mg/kg willbe effective; and in many situations, lesser doses of from about 0.5 toabout 50 mg/kg will be effective. A compound of the present inventioncan be administered in a single dose, but in the known manner ofantibacterial therapy, a compound of the present invention is typicallyadministered repeatedly over a period of time, such as a matter of daysor weeks, to ensure control of the bacterial infection.

Also in accordance with known antibacterial therapy, a compound of thepresent invention is typically formulated for convenient delivery of therequisite dose. Therefore, in another embodiment, the present inventionis directed to a pharmaceutical formulation comprising a compound of thepresent invention, in combination with a pharmaceutically-acceptablecarrier. Such carriers are well known for both oral and parenteralroutes of delivery. In general, a formulation will comprise a compoundof the present invention in a concentration of from about 0.1 to about90% by weight, and often from about 1.0 to about 3%.

The antibacterial efficacy of the present compounds is illustrated byTable II. The minimal inhibitory concentrations (MICs) were determinedusing a standard broth micro-dilution assay.

                                      TABLE II    __________________________________________________________________________    Antibacterial Activity, Minimal Inhibitory    Concentration (MIC) Against Various Organisms*    __________________________________________________________________________    Ex            SA SA  SA SH  SH  SE SPY SPN    # RESISTANT            SENSITIVE                  446                     489 447                            105 415 270                                       C203                                           P1    __________________________________________________________________________     1      >128  16    8  4   8  64  >64 8  2   2     2      128   3.4   2  2   2  8   16  4  0.125                                           0.25     3      16    1.7   4  2   1  2   2   1  ≦.06     4      14    4     2  0.125                         1  2   1   0.5                                       ≦0.06                                           0.125     5      >128  9.5   8  >64 8  64  >64 32 0.5 1     6      128   5     2  16  4  4   8   64 ≦0.06                                           ≦0.06     7      19    3     4  2   1  4   2   0.5                                       0.25                                           ≦0.06     8      >128  8     2  2   4  16  64  8     9      >128  21    8  4   8  32  32  16    10      9.5   1.7   4  2   2  1   2   2    11      38    2.6   4  2   2  1   2   2    12      128   3.5   4  1   1  2   4   1  ≦0.06                                           ≦0.06    13      >128  3.5   4  2   2  4   8   2  ≦0.06                                           ≦0.06    14      >128  3.5   2  2   4  16  32  4  ≦0.06                                           0.25    15      >128  24    8  4   16 >64 >64 16 0.25                                           0.25    16      >128  7     1  0.5 1  8   64  4  ≦0.06                                           0.125    17      >128  6.1   2  1   1  4   32  2  0.25                                           ≦0.06    18      4.7   1.7   2  2   2  2   2   2  0.25                                           2    19      19    2.6   2  2   2  2   4   2  0.25                                           2    20      9.5   5.6   4  2   2  2   2   1  ≦0.06                                           4    21      32    2.6    22      6.7   2.6   2  1   1  1   2   0.5                                       ≦.06                                           ≦.06    23      >128  5.3   4  1   4  0.5 64  4    24      16    0.87  2  1   1  0.25                                1   1    __________________________________________________________________________    ABBREVIATIONS             ORGANISM    RESISTANT             Enterococcus faecium and faecalis             (geometric mean of 4-6 isolates)    SENSITIVE             Enterococcus faecium and faecalis             (geometric mean of 4-6 isolates)    SA446    Staphylococcus aureus 446    SA489    Staphylococcus aureus 489    SA447    Staphylococcus aureus 447    SH 105   Staphylococcus haemolyticus 105    SH 415   Staphylococcus haemolyticus 415    SE 270   Staphylococcus epidermidis 270    SPY C203 Streptococcus pyogenes C203    SPN P1   Streptococcus pneumoniae P1

The N^(LEU) -thiocarbamoyl compounds of the present invention can alsobe employed as starting materials to other antibacterial compounds. Thisuse is illustrated by the following reaction sequence: ##STR4## Thus, apresent compound is treated with an organic acid, preferablytrifluoroacetic acid, in a non-polar solvent, and at a temperature offrom about 0° C. to 35° C. This treatment, the second step of an Edmandegradation, results in the loss of the leucine group including thethiocarbamoyl substituent. The resulting "hexapeptides" exhibitantibacterial activity and can be employed as described above for thepresent compounds.

The hexapeptide can thereafter be reductively alkylated to introduce analkyl group on the amine freed up by the preceding process, the "N¹ " aamine. Alkylation is achieved by reacting the hexapeptide with analdehyde to form a Schiff's base, which is then reduced to obtain the N¹-alkylhexapeptide. Both reactions are carried out in a polar solvent,such as DMF, and at temperatures of 0-100° C., preferably 60-70° C. Thepreferred reducing agent is sodium cyanoborohydride. In one embodiment,the reducing agent is added at the same time as the hexapeptide andaldehyde. The resulting N¹ -alkylated hexapeptides are useful asantibacterials and can be employed as described above for compounds ofthe present invention.

I claim:
 1. A compound of the formula ##STR5## wherein R¹ represents Oor S; R² representsalkyl of C₁ -C₁₀, phenyl, naphthyl, or phenylsubstituted by one or two substituents, each of which is independentlyhalo, loweralkyl of C₁ -C₄, loweralkoxy of C₁ -C₄, benzyloxy, nitro, or##STR6## wherein each R^(2a) is independently loweralkyl of C₁ -C₄ ; andR³ represents hydrogen or --CH₂ --R^(3a) wherein R^(3a) represents alkylof C₁ -C₁₁, alkyl of C₁ -C₁₁ --R⁴, or R⁴ -(0.sub.(0 or 1) --R⁴)₀ or 1,wherein each R⁴ is independently phenyl or phenyl substituted by one ortwo substituents, each of which is independently halo, loweralkyl of C₁-C₄, loweralkoxy of C₁ -C₄, or loweralkylthio of C₁ -C₄, or apharmaceutically acceptable salt thereof.
 2. A compound of claim 1 inwhich R¹ is S.
 3. A compound of claim 1 in which R² is phenyl.
 4. ACompound of claim 1 in which R³ is --CH₂ --R^(3a).
 5. A compound ofclaim 1 in which R³ is p-(p-chlorophenyl)benzyl.
 6. A pharmaceuticalformulation comprising a compound of claim 1 in combination with apharmaceutically-acceptable diluent or carrier.
 7. A method of treatinga bacterial infection in a host comprising the step of administering tothe host an effective amount of a formulation of claim
 6. 8. A method ofclaim 7 wherein the bacterial infection is attributable to avancomycin-resistant-enterococcus.
 9. A process for the preparation of acompound as claimed in claim 1 which comprises reacting a parentglycopeptide of the formula ##STR7## wherein R³ is as defined in claim1, with an isocyanate or isothiocyanate of the formula R¹ CN--R² whereinR¹ and R² are as defined in claim 1, and optionally forming apharmaceutically acceptable salt.